CN114395574B

CN114395574B - Porcine epidemic diarrhea virus fusion protein, and encoding gene and application thereof

Info

Publication number: CN114395574B
Application number: CN202210056292.5A
Authority: CN
Inventors: 张大生
Original assignee: Changsha Axybio Biotechnology Co ltd
Current assignee: Changsha Axybio Biotechnology Co ltd
Priority date: 2022-01-18
Filing date: 2022-01-18
Publication date: 2023-08-11
Anticipated expiration: 2042-01-18
Also published as: CN114395574A

Abstract

The invention relates to a porcine epidemic diarrhea virus fusion gene and a coding protein and application thereof, wherein the porcine CTLA4 gene and the porcine epidemic diarrhea virus S gene are selected to be synthesized into fusion genes CTLA 4-PEDV; cloning the fusion gene onto adenovirus shuttle vector plasmid, co-transfecting cells with adenovirus packaging plasmid to obtain recombinant adenovirus vector rAD-CTLA4-PEDV, and the vector can be safely used after purification and can stimulate stronger cell and humoral immune response; the recombinant adenovirus type 5 rAD-CTLA4-PEDV has good safety and high immune titer, is suitable for large-scale production after immunization for one half year, can effectively prevent and treat porcine epidemic diarrhea virus, does not generate other proteins and antibodies such as M protein of the epidemic diarrhea virus, and is easy to distinguish infected animals.

Description

Porcine epidemic diarrhea virus fusion protein, and encoding gene and application thereof

Technical Field

The invention belongs to the technical field of biotechnology, and particularly relates to a porcine epidemic diarrhea virus fusion gene and a coding protein and application thereof.

Background

Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is a high-contact intestinal infectious disease characterized by porcine diarrhea, vomiting, dehydration and high mortality in piglets of all ages caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) of the coronaviridae family. PED is widely popular in various regions of the world, and economic losses are significant. The length of the epidemic diarrhea virus S gene is different, and the length of the gene is 4143bp-4161 bp. For many years, molecular epidemiological data about the S gene of PEDV have been studied more, and it has been found through studies that the S gene can be used as a basis for PEDV genotyping, and can be divided into 2 large gene groups, G1 gene group and G2 gene group, each of which can be subdivided into 2 small genetic branches, i.e., G1-1, G1-2 and G2-1, G2-2. It was also found that the G1 gene group had a base deletion or insertion (mainly gene insertion). The G1 gene group PEDV strain has amino acid insertion and deletion phenomena besides a large number of amino acid point mutations; however, the G2 gene group PEDV strain does not have the phenomenon of amino acid insertion and mainly comprises amino acid point mutation. The amino acid sequence of the PEDV strain of the G1 gene group has 93.0% -93.8% homology with CV777 (the G2 gene group represents the strain). Since PEDV has only one serotype, amino acid variation does not affect the serotype, but is presumed to be closely related to the recommended use of the vaccine. Analysis of the S gene sequence of the new epidemic strain of PEDV revealed that the S gene of the epidemic strain of PEDV had been deleted (197 aa) and inserted (4 aa), indicating that the evolution trend of the PEDV genome increased with the use of the vaccine. Drawing the genetic evolution tree of PEDV with S gene found that PEDV can be divided into 2 distinct branches G1 and G2, the G1 branch being mainly live vaccine strains such as CV777, DR13, etc. and PEDV early isolates, while the G2 branch being a new epidemic strain of PEDV, is mainly popular in korea, usa, etc.

Currently, epidemic diarrhea vaccines mainly comprise live vaccines, inactivated vaccines, subunit vaccines and the like. However, the shown immune effect is common to subunit vaccine and inactivated vaccine, and classical live vaccine can not protect the damage caused by variant virus. We sequenced several strains of epidemic diarrhea virus S genes since 2018, found that the dominant strain S gene and CV777 were very low in amino acid homology (after multiple farms use immune CV777 live vaccines, they still developed, indicating that CV777 could not protect infection by the new strain), and AJ1102 was only 88.1% homologous, and XJDB2 was 98.2% homologous. The G2 branch of the epidemic diarrhea virus in recent years has the more and more harm to pig farms in China, and the more intensive the pig farms in China are, the more the potential threat caused by the transmission of new strains is. The classical live vaccine is safe but has poor effect, and whether the use of the new live vaccine is easy to cause strain variation and toxin expelling to cause wider infection or not has insufficient data support. It is therefore a major topic to find a safe and effective diarrhea vaccine to control the potential hazard of epidemic diarrhea viruses to pig farms.

For this reason, this patent is filed.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a porcine epidemic diarrhea virus fusion gene, and a coding protein and application thereof, and the recombinant adenovirus 5 rAD-CTLA4-PEDV has the advantages of good safety and high immune titer, is suitable for large-scale production, and can effectively prevent and treat porcine epidemic diarrhea virus.

The invention aims to provide a porcine epidemic diarrhea virus fusion gene.

Another object of the present invention is to provide a protein encoded by the porcine epidemic diarrhea virus fusion gene.

It is still another object of the present invention to provide a recombinant vector comprising the porcine epidemic diarrhea virus fusion gene as described above.

The nucleotide sequence of the porcine epidemic diarrhea virus fusion gene according to the specific embodiment of the invention is shown in SEQ ID NO. 1:

ATGGAAATGAAAGGGATGCACGTGGCCCAACCTGCAGTAGTGCTGGCCAACAGCCGGGGTGTTGCCAGCTTTGTGTGTGAGTATGGGTCTGCAGGCAAAGCTGCCGAGGTCCGGGTGACAGTGCTGCGGCGGGCCGGCAGCCAGATGACTGAAGTCTGTGCCGCGACATATACTGTGGAGGATGAGTTGACCTTCCTTGATGACTCTACATGCACTGGCACCTCCACCGAAAACAAAGTGAACCTCACCATCCAAGGGCTGAGAGCCGTGGACACTGGGCTCTACATCTGCAAGGTGGAGCTCCTGTACCCACCACCCTACTATGTGGGTATGGGCAACGGGACCCAGATTTATGTCATTGATCCAGAACCATGCCCAGATTCTGATGGTGGCGGTGGCTCGGGCGGTGGTGGATCTGGTGGCGGCGGATCTGGAACTTCCATTCAGCGTATTCTTTATTGTGATGATCCTGTTAGCCAACTCAAGTGTTCTCAGGTTGCTTTTGACCTTGACGATGGTTTTTACCCTATTTCTTCTAGAAACCTTCTGAGTCATGAACAGCCAATTTCTTTTGTTACTCTGCCATCATTTAATGATCATTCTTTTGTTAACATTACTGTCTCTGCTTCCTTTGGTGGTCATAGTGGTGCCAACCTTATTGCATCTGATACTACCATCAATGGGCTTAGTTCTTTCTGTGTTGACACTAGACAATTTACCATTTCACTGTTTTATAACGTTACAAACAGTTATGGTTATGTGTCTAAATCACAGGACAGTAATTGCCCTTTCACCTTGCAATCTGTTAATGATTACCTGTCTTTTAGCAAATTTTGTGTTTCCACCAGCCTTTTGGCTAGTGCCTGTACCATAGATCTTTTTGGTTATCCTGAGTTTGGTAGTGGTGTTAAGTTTACGTCCCTTTACTTTCAATTCACAAAGGGTGAGTTGATTACTGGCACGCCTAAACCACTTGAAGGTGTCACTGACGTTTCTTTTATGACTCTGGATGTGTGCACCAAGTATACTATCTATGGCTTTAAAGGTGAGGGTATCATTACCCTTACAAATTCTAGCATTTTGGCAGGTGTTTATTACACATCTGATTCTGGACAGTTGTTAGCTTTTAAGAATGTCACTAGTGGTGCTGTTTATTCTGTCACGCCATGTTCTTTTTCAGAGCAGGCTGCATATGTTGATGATGATATAGTGGGTGTTATTTCTAGTTTGTCTAGCTCCACTTTTAACAGTACTAGGGAGTTGCCTGGTTTCTTCTACCATTCTAATGATGGCTCTAATTGTACAGAGCCTGTGTTGGTGTATAGTAACATAGGTGTTTGTAAATCTGGCAGTATTGGCTACGTCCCATCTCAGTCTGGCCAAGTCAAAATTGCACCCATGGTTACTGGGAATATCAGTATTCCCACCAACTTTAGTATGAGTATTAGGACAGAATATTTACAGCTTTACAACACGCCTGTTAGTGTTGATTGTGCCACATATGTTTGTAATGGTAACTCTCGTTGTAAACAATTACTCACCCAGTACACTGCAGCATGTAAGACCATAGAATCAGTATTACAACTCAGCGCTAGGCTTGAGTCTGTTGAAGTTAACTCTATGCTCACTATTTCTGAAGAGGCTCTACAGTTAGCTACCATTAGTTCGTTTAATGGTGATGGATATAATTTTACTAATGTGCTGGGTGTTTCTGTGTATGATCCTGCAAGTGGCAGGGTG

the protein coded by the porcine epidemic diarrhea virus fusion gene according to the specific embodiment of the invention has the amino acid sequence shown in SEQ ID NO. 2:

MEMKGMHVAQPAVVLANSRGVASFVCEYGSAGKAAEVRVTVLRRAGSQMTEVCAATYTVEDELTFLDDSTCTGTSTENKVNLTIQGLRAVDTGLYICKVELLYPPPYYVGMGNGTQIYVIDPEPCPDSDGGGGSGGGGSGGGGSGTSIQRILYCDDPVSQLKCSQVAFDLDDGFYPISSRNLLSHEQPISFVTLPSFNDHSFVNITVSASFGGHSGANLIASDTTINGLSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGVTDVSFMTLDVCTKYTIYGFKGEGIITLTNSSILAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVDDDIVGVISSLSSSTFNSTRELPGFFYHSNDGSNCTEPVLVYSNIGVCKSGSIGYVPSQSGQVKIAPMVTGNISIPTNFSMSIRTEYLQLYNTPVSVDCATYVCNGNSRCKQLLTQYTAACKTIESVLQLSARLESVEVNSMLTISEEALQLATISSFNGDGYNFTNVLGVSVYDPASGRV

according to a specific embodiment of the present invention, the porcine epidemic diarrhea virus fusion gene (CTLA 4-PEDV) comprises a porcine CTLA4 gene and an porcine epidemic diarrhea virus S gene (PEDV). The nucleotide sequence of the S gene of the epidemic diarrhea virus is shown in SEQ ID NO. 3:

ATGGAAGGAACTTCCATTCAGCGTATTCTTTATTGTGATGATCCTGTTAGCCAACTCAAGTGTTCTCAGGTTGCTTTTGACCTTGACGATGGTTTTTACCCTATTTCTTCTAGAAACCTTCTGAGTCATGAACAGCCAATTTCTTTTGTTACTCTGCCATCATTTAATGATCATTCTTTTGTTAACATTACTGTCTCTGCTTCCTTTGGTGGTCATAGTGGTGCCAACCTTATTGCATCTGATACTACCATCAATGGGCTTAGTTCTTTCTGTGTTGACACTAGACAATTTACCATTTCACTGTTTTATAACGTTACAAACAGTTATGGTTATGTGTCTAAATCACAGGACAGTAATTGCCCTTTCACCTTGCAATCTGTTAATGATTACCTGTCTTTTAGCAAATTTTGTGTTTCCACCAGCCTTTTGGCTAGTGCCTGTACCATAGATCTTTTTGGTTATCCTGAGTTTGGTAGTGGTGTTAAGTTTACGTCCCTTTACTTTCAATTCACAAAGGGTGAGTTGATTACTGGCACGCCTAAACCACTTGAAGGTGTCACTGACGTTTCTTTTATGACTCTGGATGTGTGCACCAAGTATACTATCTATGGCTTTAAAGGTGAGGGTATCATTACCCTTACAAATTCTAGCATTTTGGCAGGTGTTTATTACACATCTGATTCTGGACAGTTGTTAGCTTTTAAGAATGTCACTAGTGGTGCTGTTTATTCTGTCACGCCATGTTCTTTTTCAGAGCAGGCTGCATATGTTGATGATGATATAGTGGGTGTTATTTCTAGTTTGTCTAGCTCCACTTTTAACAGTACTAGGGAGTTGCCTGGTTTCTTCTACCATTCTAATGATGGCTCTAATTGTACAGAGCCTGTGTTGGTGTATAGTAACATAGGTGTTTGTAAATCTGGCAGTATTGGCTACGTCCCATCTCAGTCTGGCCAAGTCAAAATTGCACCCATGGTTACTGGGAATATCAGTATTCCCACCAACTTTAGTATGAGTATTAGGACAGAATATTTACAGCTTTACAACACGCCTGTTAGTGTTGATTGTGCCACATATGTTTGTAATGGTAACTCTCGTTGTAAACAATTACTCACCCAGTACACTGCAGCATGTAAGACCATAGAATCAGTATTACAACTCAGCGCTAGGCTTGAGTCTGTTGAAGTTAACTCTATGCTCACTATTTCTGAAGAGGCTCTACAGTTAGCTACCATTAGTTCGTTTAATGGTGATGGATATAATTTTACTAATGTGCTGGGTGTTTCTGTGTATGATCCTGCAAGTGGCAGGGTG

the amino acid sequence of the protein coded by the S gene of the epidemic diarrhea virus is shown in SEQ ID NO. 4:

MEGTSIQRILYCDDPVSQLKCSQVAFDLDDGFYPISSRNLLSHEQPISFVTLPSFNDHSFVNITVSASFGGHSGANLIASDTTINGLSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGVTDVSFMTLDVCTKYTIYGFKGEGIITLTNSSILAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVDDDIVGVISSLSSSTFNSTRELPGFFYHSNDGSNCTEPVLVYSNIGVCKSGSIGYVPSQSGQVKIAPMVTGNISIPTNFSMSIRTEYLQLYNTPVSVDCATYVCNGNSRCKQLLTQYTAACKTIESVLQLSARLESVEVNSMLTISEEALQLATISSFNGDGYNFTNVLGVSVYDPASGRV

the nucleotide sequence of the CTLA4 gene of the pig is shown in SEQ ID NO. 5:

ATGAAAGGGATGCACGTGGCCCAACCTGCAGTAGTGCTGGCCAACAGCCGGGGTGTTGCCAGCTTTGTGTGTGAGTATGGGTCTGCAGGCAAAGCTGCCGAGGTCCGGGTGACAGTGCTGCGGCGGGCCGGCAGCCAGATGACTGAAGTCTGTGCCGCGACATATACTGTGGAGGATGAGTTGACCTTCCTTGATGACTCTACATGCACTGGCACCTCCACCGAAAACAAAGTGAACCTCACCATCCAAGGGCTGAGAGCCGTGGACACTGGGCTCTACATCTGCAAGGTGGAGCTCCTGTACCCACCACCCTACTATGTGGGTATGGGCAACGGGACCCAGATTTATGTCATTGATCCAGAACCATGCCCAGATTCTGAT

the invention connects the CTLA4 gene of the pig and the gene corresponding to 455-890 amino acids of the dominant strain S gene in the G2 branch of the latest separated epidemic diarrhea virus into a fusion gene. The sequence of the connected fusion gene is shown in SEQ ID NO. 6: GGTGGCGGTGGCTCGGGCGGTGGTGGATCTGGTGGCGGCGGATCT.

According to the recombinant vector of the specific embodiment of the invention, the recombinant vector contains the porcine epidemic diarrhea virus fusion gene. The recombinant vector is recombinant adenovirus vector rAD-CTLA 4-PEDV.

Preferably, the recombinant vector is a recombinant adenovirus type 5 vector.

A method for preparing a recombinant vector according to an embodiment of the present invention, the method comprising the steps of:

(1) Artificially synthesizing a porcine CTLA4 gene and a porcine epidemic diarrhea virus S gene fusion gene CTLA 4-PEDV;

(2) Subcloning the fusion gene CTLA4-PEDV synthesized in the step (1) onto an adenovirus shuttle vector plasmid P to obtain an adenovirus shuttle plasmid P-CTLA 4-PEDV;

(3) Co-transfecting the adenovirus shuttle plasmid p-CTLA4-PEDV obtained in the step (2) with an adenovirus packaging vector to a host cell;

(4) Collecting cell culture supernatant and cell precipitation lysis supernatant to obtain rough extract of CTLA4-PEDV recombinant adenovirus vector.

Specifically, in step (2), the adenovirus shuttle vector plasmid is pDC316.

More specifically, the CTLA4-PEDV fusion gene was cloned into EcoRI and HindIII cleavage sites on the pDC316 vector, resulting in the adenovirus shuttle plasmid pDC316-CTLA 4-PEDV.

Specifically, in the step (3), the adenovirus packaging vector is pBHGlox (delta) E13Cre.

Specifically, in step (3), the transfected host cell is a 293 series cell.

Preferably, the transfected host cell is a HEK293 cell.

The invention also provides a recombinant host strain containing the recombinant vector.

The invention also provides application of the recombinant vector in preparation of epidemic diarrhea vaccine or medicine.

The research shows that the adenovirus type 5 vector can generate good antibodies through injection and oral administration, and the recombinant adenovirus type 5 vector is a safe and effective gene vaccine delivery vector, and plays a great role in resisting new coronal epidemic situation of human beings. In vaccine immunization, a good immune adjuvant such as a CTLA4 gene of a pig can play a very important role in enhancing cellular immunity and humoral immunity. Based on the gene, the fusion gene formed by connecting the CTLA4 gene of the pig and the gene corresponding to 455-890 amino acids of the dominant strain S gene in the G2 branch of the latest separated epidemic diarrhea virus is cloned into an adenovirus shuttle vector, and the adenovirus shuttle vector is packaged into a recombinant adenovirus type 5 vector. The results of the study showed that recombinant adenoviruses comprising CTLA4-PEDV fusion genes produced higher levels of antibodies than recombinant adenoviruses comprising PEDV gene alone.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention firstly combines fusion genes CTLA4-PEDV composed of a pig CTLA4 gene and a pig epidemic diarrhea virus S gene; packaging into recombinant adenovirus vector, which can be safely used after purification, can stimulate stronger cell and humoral immune response, and achieves the expected purpose;

(2) Experiments show that the recombinant 5-type adenovirus vector rAD-CTLA4-PEDV containing the CTLA4-PEDV fusion gene and the recombinant 5-type adenovirus vector rAD-PEDV not containing the CTLA4 gene can stimulate the organism to generate specific humoral immunity and cellular immunity response, but the cellular immunity and humoral immunity response level of the rAD-CTLA4-PEDV is obviously higher than that of the rAD-PEDV, and the effect of fusion protein consisting of a gene adjuvant and a target gene is obviously better than that of a single target protein.

(3) The recombinant adenovirus type 5 rAD-CTLA4-PEDV has good safety and high immune titer, is suitable for large-scale production after immunization for one half year, can effectively prevent and treat porcine epidemic diarrhea virus, does not generate other proteins and antibodies such as M protein of the epidemic diarrhea virus, and is easy to distinguish infected animals.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows a map of pDC316-CTLA4-PEDV plasmid;

FIG. 2 shows the recovery of PEDV double-digested product, wherein M (DNA MARKER): 100 250, 500, 750, 1000, 2000; PEDV fragment;

FIG. 3 shows a pDC316-PEDVS plasmid map;

FIG. 4 shows a standard curve of PEDV fluorescence quantitative PCR;

FIG. 5 shows the Western-Blotting results of the target protein. 1: rAd-Null group; 2: rAd-PEDVS group; 3: rAd-CTLA4-PEDVS group; 4: rAd-CTLA 4-PEDV-1 group; 5: vaccine immune group serum for epidemic diarrhea;

FIG. 6 shows a plasmid map of pDC316-CTLA 4-PEDV-1.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.

Experimental materials

Genes, vectors and cells: a recombinant adenovirus packaging vector system, a pDC316 shuttle plasmid and an adenovirus backbone vector pBHGlox (delta) E13Cre; HEK293 cells, E.coli DH 5. Alpha. Were stored by Changsha cobra Biotechnology Co.Ltd.

The main reagent comprises: ecoRI, hindIII, T4 DNA ligase, taq DNA polymerase, available from TaKaRa; transfection reagent Lipo2000 was purchased from Invitrogen company. DMEM/High Glucose cell culture medium; pancreatin solutions were purchased from Hyclone company; fetal bovine serum FBS was purchased from Gibco company; penicillin/streptomycin solutions were purchased from Gibco company. The rest reagents are imported or homemade analytically pure.

In some more specific embodiments, the method for preparing a recombinant vector comprises the steps of:

(1) Artificially synthesizing a fusion gene CTLA4-PEDV of a swine CTLA4 gene and a swine epidemic diarrhea virus S gene (the nucleotide sequence of which is shown as SEQ ID NO. 3);

(4) Collecting cell culture supernatant and cell precipitation lysis supernatant to obtain crude CTLA4-PEDV recombinant adenovirus extract.

Specifically, in step (2), the adenovirus shuttle vector plasmid is pDC316.

Specifically, in step (3), the transfected host cell is a HEK293 cell.

More specific examples are as follows.

EXAMPLE 1 construction and protein expression of pDC316-CTLA4-PEDV plasmid pDC316-PEDV

1.1 fusion genes

(1) Artificially synthesizing a fusion gene CTLA4-PEDV of a swine CTLA4 gene and a swine epidemic diarrhea virus S gene (the nucleotide sequence is shown as SEQ ID NO.3, and a fragment of 450 th-890 th amino acid of a conserved region of the strain S gene is adopted); synthetic CTLA4-PEDV fusion gene (shown as SEQ ID NO. 1);

(2) Synthetic fusion gene CTLA 4-PEDV-1 of swine CTLA4 gene and swine epidemic diarrhea virus S gene (fragment of 493-708 amino acids of strain S gene is adopted);

the nucleotide sequence of the fusion gene CTLA 4-PEDV-1 is shown in SEQ ID NO. 7:

ATGGAAATGAAAGGGATGCACGTGGCCCAACCTGCAGTAGTGCTGGCCAACAGCCGGGGTGTTGCCAGCTTTGTGTGTGAGTATGGGTCTGCAGGCAAAGCTGCCGAGGTCCGGGTGACAGTGCTGCGGCGGGCCGGCAGCCAGATGACTGAAGTCTGTGCCGCGACATATACTGTGGAGGATGAGTTGACCTTCCTTGATGACTCTACATGCACTGGCACCTCCACCGAAAACAAAGTGAACCTCACCATCCAAGGGCTGAGAGCCGTGGACACTGGGCTCTACATCTGCAAGGTGGAGCTCCTGTACCCACCACCCTACTATGTGGGTATGGGCAACGGGACCCAGATTTATGTCATTGATCCAGAACCATGCCCAGATTCTGATGGTGGCGGTGGCTCGGGCGGTGGTGGATCTGGTGGCGGCGGATCTCAGCGTATTCTTTATTGTGATGATCCTGTTAGCCAACTCAAGTGTTCTCAGGTTGCTTTTGACCTTGACGATGGTTTTTACCCTATTTCTTCTAGAAACCTTCTGAGTCATGAACAGCCAATTTCTTTTGTTACTCTGCCATCATTTAATGATCATTCTTTTGTTAACATTACTGTCTCTGCTTCCTTTGGTGGTCATAGTGGTGCCAACCTTATTGCATCTGATACTACCATCAATGGGCTTAGTTCTTTCTGTGTTGACACTAGACAATTTACCATTTCACTGTTTTATAACGTTACAAACAGTTATGGTTATGTGTCTAAATCACAGGACAGTAATTGCCCTTTCACCTTGCAATCTGTTAATGATTACCTGTCTTTTAGCAAATTTTGTGTTTCCACCAGCCTTTTGGCTAGTGCCTGTACCATAGATCTTTTTGGTTATCCTGAGTTTGGTAGTGGTGTTAAGTTTACGTCCCTTTACTTTCAATTCACAAAGGGTGAGTTGATTACTGGCACGCCTAAACCACTTGAAGGTGTCACTGACGTTTCTTTTATGACTCTGGATGTGTGCACCAAGTATACTATCTATGGCTTTAAAGGTGAGGGTATCATTACCCTTACAAATTCT

(3) Artificially synthesizing fusion genes CTLA4-PEDVS-2 of a swine CTLA4 gene and a swine epidemic diarrhea virus S gene (adopting a full-length S protein fragment corresponding to a strain S gene);

1.2 cloning of CTLA 4-PEDV-1, CTLA 4-PEDV-2 fusion genes into EcoRI and HindIII cleavage sites on the pDC316 vector, respectively, transformation into Top10 E.coli, cloning of which was designated pDC316-CTLA4-PEDV (FIG. 1), pDC316-CTLA 4-PEDV-1 (FIG. 6), pDC316-CTLA 4-PEDV-2.

1.3 amplification of the PEDV Gene in the pDC316-CTLA4-PEDV plasmid with primer F/R.

F：5'-CGTGAATTCCACCATGGAAGGAACTTCCATTCAGCGTATTC-3'，

R：5'-CGGAAGCTTCACACCCTGCCACTTGCAGGAT-3'。

The PCR product was digested with EcoRI and HindIII, the digested product was recovered, and the recovered product was subjected to 1.0% agarose gel electrophoresis, the results of which are shown in FIG. 2.

Subcloning into EcoRI and HindIII cleavage sites in the pDC316 vector, transformation into Top10 E.coli, the positive clone was designated pDC316-PEDVS (see FIG. 3).

1.4pDC316-CTLA4-PEDV, pDC316-CTLA 4-PEDV-1, pDC316-CTLA 4-PEDV-2, pDC316-PEDV and pBHGlox (delta) E13Cre plasmid extraction

And (3) extracting plasmids by using a medium extraction kit, quantitatively determining the extracted plasmids, and carrying out the next test when the transfection requirement is met.

1.5 eukaryotic expression of fusion proteins

(1) Inoculating 1×10 in 10cm Petri dishes ⁶ HEK293 cells are cultured until the cells grow to 80-90% of the bottom of the dish;

(2) Transfection was performed using lipo2000 liposomes: pDC316-CTLA4-PEDV, pDC316-CTLA 4-PEDV-1, pDC316-CTLA 4-PEDV-2, pDC316-PEDV plasmid (pDC 316-RFP can generate red fluorescence, and can be used as positive control to monitor transfection efficiency) was taken out from 5. Mu.g to 0.5mL of serum-free DMEM medium, and 10. Mu.L lipo2000 was added to 0.5mL of serum-free DMEM medium and left standing at room temperature for 10min;

(3) Respectively dropwise adding the plasmid-containing solution in the step (2) into the lipo 2000-containing solution, uniformly mixing, and standing at room temperature for 15min;

(4) Adding the solution obtained in the step (3) into HEK293 cells, and continuously culturing the HEK293 cells by replacing a DMEM complete medium containing 10% FBS for 6 hours;

(5) HEK293 cells of the pDC316-RFP transfected group were observed for red fluorescence. 48 hours after transfection, the pDC316-RFP group was able to see very strong red fluorescence. The transfection effect is very good;

(6) Cell supernatants (containing eukaryotic expressed fusion proteins) from the plasmid transfection sets of pDC316-CTLA4-PEDV, pDC316-CTLA 4-PEDV-1, pDC316-CTLA 4-PEDV-2, pDC316-PEDV were collected separately for use.

EXAMPLE 2 packaging of recombinant adenovirus 5 rAd-CTLA4-PEDV, rAd-CTLA 4-PEDV-1 and rAd-PEDV

During the packaging of recombinant viruses, it was found that the full-length fragment of the S protein was difficult to detoxify (probably because the fragment was too large), and therefore the full-length fragment was unsuitable for use as a recombinant adenovirus vector.

2.1 packaging recombinant adenovirus 5

(1) 1X 10 seeds were grown in 10cm dishes ⁶ HEK293 cells are cultured until the cell confluence plate bottom is 80-90%;

(2) Transfection was performed using lipo2000 liposomes: pDC316-CTLA4-PEDV, pDC316-CTLA 4-PEDV-1, pDC316-PEDV plasmid 1. Mu.g and 3. Mu.g of the plasmid pBHGlox (delta) E13Cre were added to 0.5mL of serum-free DMEM medium, respectively, and 10. Mu.L of lipo2000 was added to 0.5mL of serum-free DMEM medium and allowed to stand at room temperature for 10min;

(3) Dropwise adding the plasmid solution obtained in the step (2) into the lipo 2000-containing solution, uniformly mixing, and standing at room temperature for 15min;

(4) Adding the solution obtained in the previous step into HEK293 cells, and continuously culturing the HEK293 cells by replacing a DMEM complete medium containing 10% FBS for 6 hours;

(5) After cell confluence, passage was performed at 1:3, medium was complete medium of 10% FBS:

after 3 days of passage, cytopathic reactions (CPE) were observed daily;

when the cells have obvious CPE phenomenon and have >50% wall removal, the cells are subjected to lysis and virus collection, and the steps are as follows:

1) Cells were collected in a 15mL clean centrifuge tube.

2) Centrifuge at 1500g for 5min.

3) 1mL of supernatant was left and the excess was discarded.

4) The freezing and thawing are rapidly repeated three times between the dry ice bath and 37 ℃.

5) Centrifuge at 2000g for 15min.

2.2 amplified culture of recombinant adenovirus (200 dishes as an example)

(1) Inoculating 3×10 in a culture dish with diameter of 10cm ⁶ Fine HEK293A cell;

(2) Passaging is performed at 1:5 after cells grow up until a total of 200 dish cells;

(3) Adding a proper amount of cell lysis supernatant containing adenovirus obtained in the previous experiment into HEK293 cells with 95% fusion degree;

(4) About 48-72 hours, when the cells have obvious CPE phenomenon, collecting the cells from cell supernatant and cell sediment respectively.

(5) After washing the cell pellet with PBS three times, adding PBS according to 1/10 volume of the cell culture supernatant to carry out cell resuspension, repeatedly freezing and thawing at-80 ℃ and 37 ℃ three times, centrifuging, and collecting the supernatant for later use.

PCR determination of recombinant adenovirus titres

The collected cell culture supernatant and cell pellet lysis supernatant were subjected to viral titer determination by fluorescent quantitative PCR. The primer sequences were as follows:

PEDVS-153F：5'-CTAGGGAGTTGCCTGGTTTCTTC-3'，

PEDVS-153R：5'-TAACCATGGGTGCAATTTTGAC-3'，

PEDVS-153PROBE：5'-FAM-ACCATTCTAATGATGGCTC-BHQ1-3'；

the reaction system: 2x PCR mix, 10. Mu.L; PEDVS-153f,1 μl; DNA, 2. Mu.L; H2O, 5. Mu.L; PEDVS-153r,1 μl; PEDV-153 PROBE, 1. Mu.L.

Reaction conditions: 95 ℃ for 3min;95 ℃,15s 40cycle;60 ℃ for 30s.

The amplification curve of the positive control after the completion of the reaction is shown in FIG. 4. And calculating a standard curve equation according to the result: y= -0.2803logx+13.211; r is R ² ＝0.9997。

TABLE 1 CT value results corresponding to fluorescent quantitative PCR for different concentration standards

The titer of the virus of interest was calculated from the standard curve. The titer of viruses in rAd-CTLA 4-PEDV-1 and rAd-PEDV cell culture supernatants was approximately 2-5X 10 ⁸ Second order/mL; the virus titer in the cell pellet lysis supernatants of rAd-CTLA4-PEDV, rAd-CTLA 4-PEDV-1 and rAd-PEDV was 5-10X 10 ⁹ Second order/mL. The difference in rAd-CTLA4-PEDV, rAd-CTLA 4-PEDV-1 and rAd-PEDV virus yields is not obvious.

EXAMPLE 3 determination of IgG levels in serum of rAd-CTLA4-PEDV, rAd-CTLA 4-PEDV-1, rAd-PEDV-injected mice

3.1 recombinant rAd-CTLA4-PEDV, rAd-CTLA 4-PEDV-1, rAd-PEDV immunized mice

On a mouse model, the immune doses of recombinant rAd-CTLA4-PEDV, rAd-CTLA 4-PEDV-1 and rAd-PEDV vaccines are studied, and IgG determination in serum is determined.

SPF-grade Kunming mice (4-6 weeks old) were purchased from Hunan Jingda laboratory animals Co. The recombinant adenovirus vector and physiological saline are prepared into vaccine, the injection volume is 50 mu L, blood is collected weekly after immunization, and the prepared serum is frozen in a refrigerator at the temperature of-70 ℃ for standby. The mice were grouped, immunized and immunized as shown in table 2:

TABLE 2 immunization groups and cases

Remarks: rAd5-Null is a recombinant virus packaged by empty vector without inserted gene. A vaccine was immunized 2 weeks after the first immunization for the 2 nd immunization.

3.2 determination of PEDV antibodies in serum by immunoblotting

(1) 100 microliters of the cell culture supernatant of 1.4 was added to the protein loading buffer.

(2) 10. Mu.l of the sample prepared in 3.1 was added to the spotted wells and subjected to 10% SDS-PAGE. Electrophoresis conditions: the electrophoresis stop time was determined by pre-staining protein markers at a constant pressure of 100V.

(3) Transferring the membrane, and transferring the protein on the gel block after electrophoresis to the PVDF membrane.

(4) Closing: the membrane was completely immersed in 5% nonfat dry milk-TBST and gently shaken at room temperature for 30min.

(5) The sera of each group (rAd 5-Null, rAd-PEDV, rAd-CTLA4-PEDV, a vaccine) collected under 3.1 item were diluted 100-fold with TBST, incubated in an antibody incubation box for 10min at room temperature, and left at 4℃overnight.

(6) The next day the film was removed from 4 ℃. Washing the film: TBST washes the membrane 5 times for 3min each.

(7) The coat Anti-Mouse IgG (H+L) -HRP secondary antibody was diluted 1:5000 with TBST, and the diluted antibody was added to the antibody incubation box and incubated at room temperature for 40min.

(8) Washing the film: TBST washes the membrane 5 times for 3min each.

(9) ECL was applied to the film and reacted for 2min, film exposure: 10s-3min (exposure time is adjusted according to different light intensities), developing for 2min, and fixing.

(10) Results of immunoblotting.

As shown in FIG. 5, the results of immunoblotting showed that the group 1 sera had no target bands, and the group 2,3,4, and 5 sera had target bands, respectively, differing in the expression of PEDV proteins. It is demonstrated that the sera of rAd-PEDV, rAd-CTLA 4-PEDV-1 injected and the epidemic diarrhea vaccine immunized all can react with eukaryotic expressed PEDV protein to generate specific antigen-antibody reaction. Because CTLA4-PEDV fusion protein is 62kD in size, western-Blotting results bands are at 62kD for each serum group.

3.3 Indirect ELISA detection of Total IgG in isolated serum samples

Coating the PEDV protein antigen prepared in the above step, a 96-well ELISA plate was coated at a concentration of 1. Mu.g/mL and 100. Mu.L per well, at 37℃for 2 hours, and then washed with PBST (PBS+Tween). Adding 100 μl of 5% skimmed milk PBST blocking solution into each well, blocking at 37deg.C for 1 hr, and washing; adding PBS diluted (1:100) immune serum to be detected into each hole, incubating for 1h at 37 ℃, and washing; HRP (horseradish peroxidase) labeled goat anti-mouse IgG diluted (1:2000) with PBS is added to each well as a secondary antibody, and incubated for 1h at 37 ℃, and washed; 100 mu L of TMB color development liquid (3, 3', 5' -tetramethyl benzidine) is added into each hole, after the color development is carried out for 10min at room temperature, 50 mu L of 2M sulfuric acid is added into each hole to terminate the reaction, and an enzyme-labeled instrument is used for measuring OD _450nm Absorbance values. And analyzing the result. When the sample OD ₄₅₀ The value is not less than (OD of negative serum) ₄₅₀ Value +3 times standardVariance) is positive.

The serum PEDVS IgG antibody detection results after 4 weeks of immunization are shown in table 3:

TABLE 3 PEDV IgG antibody levels in mouse serum after 4 weeks of immunization

As can be seen from Table 3, rAd5-Null group did not produce specific PEDV IgG antibodies; higher PEDV IgG antibodies were detected in the rAD-PEDV group, rAD-CTLA 4-PEDV-1 group and in the serum of the epidemic diarrhea vaccine immunized group, wherein the antibody levels of the serum of the rAD-CTLA4-PEDV group and the serum of the epidemic diarrhea vaccine immunized group are significantly higher than those of the serum of the rAD5-PEDV group, the serum of the rAD-CTLA 4-PEDV-1 group and the serum of the rAD-CTLA4-PEDV group and the serum of the epidemic diarrhea vaccine immunized group are not significantly different, which indicates that the primary immunization of the rAD-CTLA4-PEDV group and the secondary immunization of a certain diarrhea vaccine produce equivalent antibody levels.

3.4 determination of IgG subtype in serum of rAd5-CTLA4-PEDV injected mice and evaluation of immune Effect

The ELISA assay results for IgG1, igG2a, igG2b, and IgG3 antibodies in serum (kit purchased from Wohan Iretto Biotechnology Co., ltd.) are shown in Table 4:

TABLE 4 detection results of IgG1, igG2a, igG2b, and IgG3 antibodies in serum

Elevated IgG1 levels indicate that the immune response in protective immunity progresses toward Th2 (humoral immune response); elevated IgG2a levels suggest that the immune response in protective immunity progresses toward Th1 (cellular immune response). As can be seen from Table 4, the IgG1, igG2a, igG2b and IgG3 antibodies were elevated in the rAD-PEDV group, rAD-CTLA 4-PEDV-1 group and certain diarrhea vaccine immunized group as compared to the rAD5-Null group; the rAD-CTLA4-PEDV group and the rAD-CTLA 4-PEDV-1 group have IgG1 lower than that of a diarrhea vaccine, but the rAD-CTLA4-PEDV group has IgG2a higher than that of a diarrhea vaccine and the rAD-CTLA 4-PEDV-1 group, and the results show that the liquid immune response level of the rAD-CTLA4-PEDV group is lower than that of a diarrhea vaccine, but the cell immune response level of the rAD-CTLA4-PEDV group is higher than that of a diarrhea vaccine. The rAd-CTLA4-PEDV of the invention can strengthen the cellular immune state of mice, thereby enhancing the cellular immune level and generating stronger cellular immune response; improving immunity and anti-infection ability.

To obtain a vaccine against the G2 branch of the newly isolated epidemic diarrhea virus, we selected fragments of amino acids 450-890, 493-708 and the full length S protein fragment corresponding to the S gene of the conserved region of the S gene of the dominant strain. During the process of packaging recombinant viruses, it was found that the full-length fragment of the S protein is difficult to be toxic (the possible reason that the fragment is too large), so that the full-length fragment is not suitable for use as a recombinant adenovirus vector; the use of the fragment of amino acids 493-708 of the S gene in fusion with the Brucella OMP16 protein in our issued patent (patent No. 201610527708) enables the production of good antibodies in pigs by oral administration using lactic acid bacteria as a carrier. However, the fragment packaged adenovirus produced higher levels of antibodies than the fusion fragment of the study, although in the same amount, probably because the lactic acid bacteria were associated with a different vector than the adenovirus. Determining a conserved region of the S gene by gene comparison; meanwhile, CTLA-4 mainly acts in the T cell activation phase of lymphoid organs, inhibits T cell overactivation, immunomodulation by competing with the T cell activating receptor CD28 for binding to the common ligands CD80 and CD86, and our fusion protein produces the expected effect through adenovirus vector mediation.

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

SEQUENCE LISTING

<110> Changsha loving cobble biotechnology Co., ltd

<120> porcine epidemic diarrhea virus fusion protein, and coding gene and application thereof

<130> 20220217

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 1740

<212> DNA

<213> Synthesis

<400> 1

atggaaatga aagggatgca cgtggcccaa cctgcagtag tgctggccaa cagccggggt 60

gttgccagct ttgtgtgtga gtatgggtct gcaggcaaag ctgccgaggt ccgggtgaca 120

gtgctgcggc gggccggcag ccagatgact gaagtctgtg ccgcgacata tactgtggag 180

gatgagttga ccttccttga tgactctaca tgcactggca cctccaccga aaacaaagtg 240

aacctcacca tccaagggct gagagccgtg gacactgggc tctacatctg caaggtggag 300

ctcctgtacc caccacccta ctatgtgggt atgggcaacg ggacccagat ttatgtcatt 360

gatccagaac catgcccaga ttctgatggt ggcggtggct cgggcggtgg tggatctggt 420

ggcggcggat ctggaacttc cattcagcgt attctttatt gtgatgatcc tgttagccaa 480

ctcaagtgtt ctcaggttgc ttttgacctt gacgatggtt tttaccctat ttcttctaga 540

aaccttctga gtcatgaaca gccaatttct tttgttactc tgccatcatt taatgatcat 600

tcttttgtta acattactgt ctctgcttcc tttggtggtc atagtggtgc caaccttatt 660

gcatctgata ctaccatcaa tgggcttagt tctttctgtg ttgacactag acaatttacc 720

atttcactgt tttataacgt tacaaacagt tatggttatg tgtctaaatc acaggacagt 780

aattgccctt tcaccttgca atctgttaat gattacctgt cttttagcaa attttgtgtt 840

tccaccagcc ttttggctag tgcctgtacc atagatcttt ttggttatcc tgagtttggt 900

agtggtgtta agtttacgtc cctttacttt caattcacaa agggtgagtt gattactggc 960

acgcctaaac cacttgaagg tgtcactgac gtttctttta tgactctgga tgtgtgcacc 1020

aagtatacta tctatggctt taaaggtgag ggtatcatta cccttacaaa ttctagcatt 1080

ttggcaggtg tttattacac atctgattct ggacagttgt tagcttttaa gaatgtcact 1140

agtggtgctg tttattctgt cacgccatgt tctttttcag agcaggctgc atatgttgat 1200

gatgatatag tgggtgttat ttctagtttg tctagctcca cttttaacag tactagggag 1260

ttgcctggtt tcttctacca ttctaatgat ggctctaatt gtacagagcc tgtgttggtg 1320

tatagtaaca taggtgtttg taaatctggc agtattggct acgtcccatc tcagtctggc 1380

caagtcaaaa ttgcacccat ggttactggg aatatcagta ttcccaccaa ctttagtatg 1440

agtattagga cagaatattt acagctttac aacacgcctg ttagtgttga ttgtgccaca 1500

tatgtttgta atggtaactc tcgttgtaaa caattactca cccagtacac tgcagcatgt 1560

aagaccatag aatcagtatt acaactcagc gctaggcttg agtctgttga agttaactct 1620

atgctcacta tttctgaaga ggctctacag ttagctacca ttagttcgtt taatggtgat 1680

ggatataatt ttactaatgt gctgggtgtt tctgtgtatg atcctgcaag tggcagggtg 1740

<210> 2

<211> 580

<212> PRT

<213> Synthesis

<400> 2

Met Glu Met Lys Gly Met His Val Ala Gln Pro Ala Val Val Leu Ala

1 5 10 15

Asn Ser Arg Gly Val Ala Ser Phe Val Cys Glu Tyr Gly Ser Ala Gly

20 25 30

Lys Ala Ala Glu Val Arg Val Thr Val Leu Arg Arg Ala Gly Ser Gln

35 40 45

Met Thr Glu Val Cys Ala Ala Thr Tyr Thr Val Glu Asp Glu Leu Thr

50 55 60

Phe Leu Asp Asp Ser Thr Cys Thr Gly Thr Ser Thr Glu Asn Lys Val

65 70 75 80

Asn Leu Thr Ile Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Ile

85 90 95

Cys Lys Val Glu Leu Leu Tyr Pro Pro Pro Tyr Tyr Val Gly Met Gly

100 105 110

Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser

115 120 125

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Thr Ser Ile Gln Arg Ile Leu Tyr Cys Asp Asp Pro Val Ser Gln

145 150 155 160

Leu Lys Cys Ser Gln Val Ala Phe Asp Leu Asp Asp Gly Phe Tyr Pro

165 170 175

Ile Ser Ser Arg Asn Leu Leu Ser His Glu Gln Pro Ile Ser Phe Val

180 185 190

Thr Leu Pro Ser Phe Asn Asp His Ser Phe Val Asn Ile Thr Val Ser

195 200 205

Ala Ser Phe Gly Gly His Ser Gly Ala Asn Leu Ile Ala Ser Asp Thr

210 215 220

Thr Ile Asn Gly Leu Ser Ser Phe Cys Val Asp Thr Arg Gln Phe Thr

225 230 235 240

Ile Ser Leu Phe Tyr Asn Val Thr Asn Ser Tyr Gly Tyr Val Ser Lys

245 250 255

Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu Gln Ser Val Asn Asp Tyr

260 265 270

Leu Ser Phe Ser Lys Phe Cys Val Ser Thr Ser Leu Leu Ala Ser Ala

275 280 285

Cys Thr Ile Asp Leu Phe Gly Tyr Pro Glu Phe Gly Ser Gly Val Lys

290 295 300

Phe Thr Ser Leu Tyr Phe Gln Phe Thr Lys Gly Glu Leu Ile Thr Gly

305 310 315 320

Thr Pro Lys Pro Leu Glu Gly Val Thr Asp Val Ser Phe Met Thr Leu

325 330 335

Asp Val Cys Thr Lys Tyr Thr Ile Tyr Gly Phe Lys Gly Glu Gly Ile

340 345 350

Ile Thr Leu Thr Asn Ser Ser Ile Leu Ala Gly Val Tyr Tyr Thr Ser

355 360 365

Asp Ser Gly Gln Leu Leu Ala Phe Lys Asn Val Thr Ser Gly Ala Val

370 375 380

Tyr Ser Val Thr Pro Cys Ser Phe Ser Glu Gln Ala Ala Tyr Val Asp

385 390 395 400

Asp Asp Ile Val Gly Val Ile Ser Ser Leu Ser Ser Ser Thr Phe Asn

405 410 415

Ser Thr Arg Glu Leu Pro Gly Phe Phe Tyr His Ser Asn Asp Gly Ser

420 425 430

Asn Cys Thr Glu Pro Val Leu Val Tyr Ser Asn Ile Gly Val Cys Lys

435 440 445

Ser Gly Ser Ile Gly Tyr Val Pro Ser Gln Ser Gly Gln Val Lys Ile

450 455 460

Ala Pro Met Val Thr Gly Asn Ile Ser Ile Pro Thr Asn Phe Ser Met

465 470 475 480

Ser Ile Arg Thr Glu Tyr Leu Gln Leu Tyr Asn Thr Pro Val Ser Val

485 490 495

Asp Cys Ala Thr Tyr Val Cys Asn Gly Asn Ser Arg Cys Lys Gln Leu

500 505 510

Leu Thr Gln Tyr Thr Ala Ala Cys Lys Thr Ile Glu Ser Val Leu Gln

515 520 525

Leu Ser Ala Arg Leu Glu Ser Val Glu Val Asn Ser Met Leu Thr Ile

530 535 540

Ser Glu Glu Ala Leu Gln Leu Ala Thr Ile Ser Ser Phe Asn Gly Asp

545 550 555 560

Gly Tyr Asn Phe Thr Asn Val Leu Gly Val Ser Val Tyr Asp Pro Ala

565 570 575

Ser Gly Arg Val

580

<210> 3

<211> 1314

<212> DNA

<213> unknown

<400> 3

atggaaggaa cttccattca gcgtattctt tattgtgatg atcctgttag ccaactcaag 60

tgttctcagg ttgcttttga ccttgacgat ggtttttacc ctatttcttc tagaaacctt 120

ctgagtcatg aacagccaat ttcttttgtt actctgccat catttaatga tcattctttt 180

gttaacatta ctgtctctgc ttcctttggt ggtcatagtg gtgccaacct tattgcatct 240

gatactacca tcaatgggct tagttctttc tgtgttgaca ctagacaatt taccatttca 300

ctgttttata acgttacaaa cagttatggt tatgtgtcta aatcacagga cagtaattgc 360

cctttcacct tgcaatctgt taatgattac ctgtctttta gcaaattttg tgtttccacc 420

agccttttgg ctagtgcctg taccatagat ctttttggtt atcctgagtt tggtagtggt 480

gttaagttta cgtcccttta ctttcaattc acaaagggtg agttgattac tggcacgcct 540

aaaccacttg aaggtgtcac tgacgtttct tttatgactc tggatgtgtg caccaagtat 600

actatctatg gctttaaagg tgagggtatc attaccctta caaattctag cattttggca 660

ggtgtttatt acacatctga ttctggacag ttgttagctt ttaagaatgt cactagtggt 720

gctgtttatt ctgtcacgcc atgttctttt tcagagcagg ctgcatatgt tgatgatgat 780

atagtgggtg ttatttctag tttgtctagc tccactttta acagtactag ggagttgcct 840

ggtttcttct accattctaa tgatggctct aattgtacag agcctgtgtt ggtgtatagt 900

aacataggtg tttgtaaatc tggcagtatt ggctacgtcc catctcagtc tggccaagtc 960

aaaattgcac ccatggttac tgggaatatc agtattccca ccaactttag tatgagtatt 1020

aggacagaat atttacagct ttacaacacg cctgttagtg ttgattgtgc cacatatgtt 1080

tgtaatggta actctcgttg taaacaatta ctcacccagt acactgcagc atgtaagacc 1140

atagaatcag tattacaact cagcgctagg cttgagtctg ttgaagttaa ctctatgctc 1200

actatttctg aagaggctct acagttagct accattagtt cgtttaatgg tgatggatat 1260

aattttacta atgtgctggg tgtttctgtg tatgatcctg caagtggcag ggtg 1314

<210> 4

<211> 438

<212> PRT

<213> unknown

<400> 4

Met Glu Gly Thr Ser Ile Gln Arg Ile Leu Tyr Cys Asp Asp Pro Val

1 5 10 15

Ser Gln Leu Lys Cys Ser Gln Val Ala Phe Asp Leu Asp Asp Gly Phe

20 25 30

Tyr Pro Ile Ser Ser Arg Asn Leu Leu Ser His Glu Gln Pro Ile Ser

35 40 45

Phe Val Thr Leu Pro Ser Phe Asn Asp His Ser Phe Val Asn Ile Thr

50 55 60

Val Ser Ala Ser Phe Gly Gly His Ser Gly Ala Asn Leu Ile Ala Ser

65 70 75 80

Asp Thr Thr Ile Asn Gly Leu Ser Ser Phe Cys Val Asp Thr Arg Gln

85 90 95

Phe Thr Ile Ser Leu Phe Tyr Asn Val Thr Asn Ser Tyr Gly Tyr Val

100 105 110

Ser Lys Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu Gln Ser Val Asn

115 120 125

Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr Ser Leu Leu Ala

130 135 140

Ser Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro Glu Phe Gly Ser Gly

145 150 155 160

Val Lys Phe Thr Ser Leu Tyr Phe Gln Phe Thr Lys Gly Glu Leu Ile

165 170 175

Thr Gly Thr Pro Lys Pro Leu Glu Gly Val Thr Asp Val Ser Phe Met

180 185 190

Thr Leu Asp Val Cys Thr Lys Tyr Thr Ile Tyr Gly Phe Lys Gly Glu

195 200 205

Gly Ile Ile Thr Leu Thr Asn Ser Ser Ile Leu Ala Gly Val Tyr Tyr

210 215 220

Thr Ser Asp Ser Gly Gln Leu Leu Ala Phe Lys Asn Val Thr Ser Gly

225 230 235 240

Ala Val Tyr Ser Val Thr Pro Cys Ser Phe Ser Glu Gln Ala Ala Tyr

245 250 255

Val Asp Asp Asp Ile Val Gly Val Ile Ser Ser Leu Ser Ser Ser Thr

260 265 270

Phe Asn Ser Thr Arg Glu Leu Pro Gly Phe Phe Tyr His Ser Asn Asp

275 280 285

Gly Ser Asn Cys Thr Glu Pro Val Leu Val Tyr Ser Asn Ile Gly Val

290 295 300

Cys Lys Ser Gly Ser Ile Gly Tyr Val Pro Ser Gln Ser Gly Gln Val

305 310 315 320

Lys Ile Ala Pro Met Val Thr Gly Asn Ile Ser Ile Pro Thr Asn Phe

325 330 335

Ser Met Ser Ile Arg Thr Glu Tyr Leu Gln Leu Tyr Asn Thr Pro Val

340 345 350

Ser Val Asp Cys Ala Thr Tyr Val Cys Asn Gly Asn Ser Arg Cys Lys

355 360 365

Gln Leu Leu Thr Gln Tyr Thr Ala Ala Cys Lys Thr Ile Glu Ser Val

370 375 380

Leu Gln Leu Ser Ala Arg Leu Glu Ser Val Glu Val Asn Ser Met Leu

385 390 395 400

Thr Ile Ser Glu Glu Ala Leu Gln Leu Ala Thr Ile Ser Ser Phe Asn

405 410 415

Gly Asp Gly Tyr Asn Phe Thr Asn Val Leu Gly Val Ser Val Tyr Asp

420 425 430

Pro Ala Ser Gly Arg Val

435

<210> 5

<211> 381

<212> DNA

<213> unknown

<400> 5

atgaaaggga tgcacgtggc ccaacctgca gtagtgctgg ccaacagccg gggtgttgcc 60

agctttgtgt gtgagtatgg gtctgcaggc aaagctgccg aggtccgggt gacagtgctg 120

cggcgggccg gcagccagat gactgaagtc tgtgccgcga catatactgt ggaggatgag 180

ttgaccttcc ttgatgactc tacatgcact ggcacctcca ccgaaaacaa agtgaacctc 240

accatccaag ggctgagagc cgtggacact gggctctaca tctgcaaggt ggagctcctg 300

tacccaccac cctactatgt gggtatgggc aacgggaccc agatttatgt cattgatcca 360

gaaccatgcc cagattctga t 381

<210> 6

<211> 45

<212> DNA

<213> unknown

<400> 6

ggtggcggtg gctcgggcgg tggtggatct ggtggcggcg gatct 45

<210> 7

<211> 1062

<212> DNA

<213> unknown

<400> 7

atggaaatga aagggatgca cgtggcccaa cctgcagtag tgctggccaa cagccggggt 60

gttgccagct ttgtgtgtga gtatgggtct gcaggcaaag ctgccgaggt ccgggtgaca 120

gtgctgcggc gggccggcag ccagatgact gaagtctgtg ccgcgacata tactgtggag 180

gatgagttga ccttccttga tgactctaca tgcactggca cctccaccga aaacaaagtg 240

aacctcacca tccaagggct gagagccgtg gacactgggc tctacatctg caaggtggag 300

ctcctgtacc caccacccta ctatgtgggt atgggcaacg ggacccagat ttatgtcatt 360

gatccagaac catgcccaga ttctgatggt ggcggtggct cgggcggtgg tggatctggt 420

ggcggcggat ctcagcgtat tctttattgt gatgatcctg ttagccaact caagtgttct 480

caggttgctt ttgaccttga cgatggtttt taccctattt cttctagaaa ccttctgagt 540

catgaacagc caatttcttt tgttactctg ccatcattta atgatcattc ttttgttaac 600

attactgtct ctgcttcctt tggtggtcat agtggtgcca accttattgc atctgatact 660

accatcaatg ggcttagttc tttctgtgtt gacactagac aatttaccat ttcactgttt 720

tataacgtta caaacagtta tggttatgtg tctaaatcac aggacagtaa ttgccctttc 780

accttgcaat ctgttaatga ttacctgtct tttagcaaat tttgtgtttc caccagcctt 840

ttggctagtg cctgtaccat agatcttttt ggttatcctg agtttggtag tggtgttaag 900

tttacgtccc tttactttca attcacaaag ggtgagttga ttactggcac gcctaaacca 960

cttgaaggtg tcactgacgt ttcttttatg actctggatg tgtgcaccaa gtatactatc 1020

tatggcttta aaggtgaggg tatcattacc cttacaaatt ct 1062

Claims

1. The porcine epidemic diarrhea virus fusion gene is characterized by comprising a porcine CTLA4 gene and an porcine epidemic diarrhea virus S gene, wherein the nucleotide sequence of the porcine epidemic diarrhea virus fusion gene is shown as SEQ ID NO. 1.

2. The protein encoded by the porcine epidemic diarrhea virus fusion gene according to claim 1, wherein the amino acid sequence of the protein encoded by the porcine epidemic diarrhea virus fusion gene is shown as SEQ ID NO. 2.

3. A recombinant vector, which contains the porcine epidemic diarrhea virus fusion gene of claim 1, and is a recombinant adenovirus type 5 vector.

4. A method of preparing the recombinant vector of claim 3, comprising the steps of:

(1) Synthesizing a porcine CTLA4 gene and a porcine epidemic diarrhea virus S gene fusion gene CTLA 4-PEDV;

(2) Cloning the fusion gene CTLA4-PEDV synthesized in the step (1) onto an adenovirus shuttle vector plasmid P to obtain an adenovirus shuttle plasmid P-CTLA 4-PEDV;

5. The method of claim 4, wherein in step (2), the adenovirus shuttle vector plasmid is pDC316.

6. The method according to claim 4, wherein in the step (3), the adenovirus packaging vector is pBHGlox (delta) E13Cre.

7. The method according to claim 4, wherein in the step (3), the transfected host cells are 293 series cells.

8. The method of claim 7, wherein the transfected host cell is HEK 293.

9. A recombinant host strain comprising the fusion gene of claim 1.

10. Use of the fusion gene of claim 1 in the preparation of an epidemic diarrhea vaccine or medicament.